The genomes of all organisms undergo spontaneous mutation in the course of their continuing evolution, generating variant forms of progenitor nucleic acid sequences (Gusella, Ann. Rev. Biochem. 55, 831-854 (1986)). The variant form may confer an evolutionary advantage or disadvantage relative to a progenitor form, or may be neutral. In some instances, a variant form confers a lethal disadvantage and is not transmitted to subsequent generations of the organism. In other instances, a variant form confers an evolutionary advantage to the species and is eventually incorporated into the DNA of many or most members of the species and effectively becomes the progenitor form. In many instances, both progenitor and variant forms survive and co-exist in a species population. The coexistence of multiple forms of a sequence gives rise to polymorphisms.
Some polymorphisms take the form of single nucleotide variations between individuals of the same species and are far more frequent than other types of polymorphisms. Some single nucleotide polymorphisms (SNPs) occur in protein-coding nucleic acid sequences (coding sequence SNP (cSNP)), in which case, one of the polymorphic forms may give rise to the expression of a defective or otherwise variant protein and, potentially, a genetic disease. Examples of genes in which polymorphisms within coding sequences give rise to genetic disease include xcex2-globin (sickle cell anemia), apoE4 (Alzheimer""s Disease), Factor V Leiden (thrombosis), and CFTR (cystic fibrosis). cSNPs can alter the codon sequence of the gene and therefore specify an alternative amino acid. Such changes are called xe2x80x9cmissensexe2x80x9d when another amino acid is substituted, and xe2x80x9cnonsensexe2x80x9d when the alternative codon specifies a stop signal in protein translation. When the cSNP does not alter the amino acid specified the cSNP is called xe2x80x9csilentxe2x80x9d. Other single nucleotide polymorphisms occur in noncoding regions. Some of these polymorphisms may also result in defective protein expression (e.g., as a result of defective splicing). Other single nucleotide polymorphisms have no phenotypic effects.
Single nucleotide polymorphisms can be used in the same manner as RFLPs and VNTRs, but offer several advantages. Single nucleotide polymorphisms occur with greater frequency and are spaced more uniformly throughout the genome than other forms of polymorphism. The greater frequency and uniformity of single nucleotide polymorphisms means that there is a greater probability that such a polymorphism will be found in close proximity to a genetic locus of interest than would be the case for other polymorphisms. The different forms of characterized single nucleotide polymorphisms are often easier to distinguish than other types of polymorphism (e.g., by use of assays employing allele-specific hybridization probes or primers).
Work described herein pertains to the identification of polymorphisms which can predispose individuals to disease, particularly cardiovascular disease, by re-sequencing the VLDLr gene in a number of individuals. SNPs in this gene were identified as described herein. For example, two SNPs were identified in the coding region (one in exon 2 and one in exon 14), fourteen SNPs were identified in introns, four SNPs were identified in the upstream regulatory region, and one SNP was identified in the 3xe2x80x2 untranslated region (UTR). Additional details regarding these SNPs are shown in Tables 1 and 2.
In one embodiment of the present invention, the invention relates to a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of the nucleic acid sequences listed in Table 2 (SEQ ID NOS: 1-22), or a portion thereof which is at least 10 nucleotides in length and comprises a polymorphic site identified in Table 2. More specifically, the nucleic acid molecule can be at least 11, 15 or 20 nucleotides in length. In a preferred embodiment, the nucleotide at the polymorphic site is a variant nucleotide; that is, the nucleotide at the polymorphic site is different from the nucleotide at the polymorphic site in a corresponding reference allele (i.e., the reference nucleotide). In one embodiment, the nucleotide at the polymorphic site for a specified nucleic acid molecule is the variant nucleotide shown in Table 2.
In another embodiment, the present invention relates to an allele-specific oligonucleotide that hybridizes to a nucleic acid molecule having a nucleic acid sequence selected from the nucleic acid sequences listed in Table 2 (SEQ ID NOS: 1-22). In a preferred embodiment, the allele-specific oligonucleotide is at least 10 nucleotides in length and includes a polymorphic site identified in Table 2. In one embodiment, the allele-specific oligonucleotide is a probe. In one embodiment, the oligonucleotide can have a central position which aligns with the polymorphic site of the nucleic acid molecule to which it hybridizes. Alternatively, the allele-specific oligonucleotide is a primer. In one embodiment, the primer can be such that the 3xe2x80x2 end of the primer aligns with the polymorphic site of the nucleic acid molecule to which it hybridizes. In preferred embodiments, the allele-specific oligonucleotide hybridizes specifically to either the reference or variant form of the nucleic acid molecules of the invention. That is, preferably the allele-specific oligonucleotide hybridizes only to a nucleic acid molecule (e.g., SEQ ID NO: 1) having the reference nucleotide at the polymorphic site and not to the corresponding nucleic acid molecule (e.g., SEQ ID NO: 1) having the variant nucleotide at the polymorphic site, or vice versa.
In another embodiment, the present invention relates to an isolated gene product encoded by a nucleic acid molecule described herein. In one embodiment, the invention relates to an isolated protein or peptide which is encoded by a nucleic acid molecule described herein. For example, the invention relates to proteins and peptides encoded by the variant form of the nucleic acid molecules described herein. In a preferred embodiment, a protein or peptide encoded by the variant form of the nucleic acid molecule(s) of the invention contains an amino acid alteration (e.g., insertion, deleteion or substitution of one or more amino acids) as compared with the protein or peptide encoded by the corresponding reference form of the nucleic acid molecule(s).
In another embodiment, the present invention is directed to a method of analyzing a nucleic acid sample for polymorphisms of the invention, comprising obtaining a nucleic acid sample from one or more individuals, and determining the nucleotide occupying one or more of the polymorphic sites of the nucleic acid molecule(s) shown in Table 2. In one embodiment, the nucleic acid sample can be obtained from a plurality of individuals, and the nucleotide occupying one or more of the polymorphic positions is determined in each of the individuals, and the method can further include testing each individual for the presence of a disease phenotype and correlating the presence of the disease phenotype with the nucleotide present at the polymorphic site(s).
In another embodiment, the present invention is directed to a method for diagnosing, aiding in the diagnosis of or predicting the likelihood that an individual will have a cardiovascular disease, comprising the steps of obtaining a nucleic acid sample from an individual to be assessed and determining the nucleotide present at a polymorphic site of the VLDLr gene shown in Table 2, such that the presence of nucleotide associated with a lower likelihood of having a cardiovascular disease indicates that the individual has a lower likelihood of having a cardiovascular disease or a greater likelihood of having reduced symptomology associated with a cardiovascular disease. In one embodiment, the cardiovascular disease is coronary heart disease. In another embodiment, the individual is an individual at risk for development of cardiovascular disease.
In another embodiment, the present invention is directed to a method for diagnosing, aiding in the diagnosis of or predicting the likelihood that an individual will have a cardiovascular disease, comprising the steps of obtaining a nucleic acid sample from an individual to be assessed and determining the nucleotide present at a polymorphic site of the VLDLr gene shown in Table 2, such that the presence of nucleotide associated with a greater likelihood of having a cardiovascular disease indicates that the individual has a greater likelihood of having a cardiovascular disease or a greater likelihood of having increased symptomology associated with a cardiovascular disease. In one embodiment, the cardiovascular disease is coronary heart disease. In another embodiment, the individual is an individual at risk for development of cardiovascular disease.